ROCK2 is critical for the enhancement of actomyosin contractility in cultured podocytes

• Main Authors: Ru-Hsuan Zheng, 鄭濡萱
• Other Authors: Hsiao-Hui Lee
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/sf55rv

碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 107 === Ultrafiltration of plasma in the renal glomeruli is a major function of the kidney. The highly differentiated podocytes have specialized foot processes (FP) that cover the outer surfaces of the glomerular capillaries and form the slit diaphragm (SD) to prevent protein loss during filtration. They adhere to the underlying glomerular basement membrane (GBM) via integrin and forms focal adhesions (FA). The connecting actin cytoskeleton is contractive to provide resistance of the network to mechanical strain caused by filtration. Previously, we found that α-actinin-4 contributes to Shp2 targeting at FA for RhoA/ROCK activation in podocytes. In this study, I further investigated the biological role of ROCK in podocytes. Using a conditional immobilized mouse podocytes as a cell model, I found that ROCK2 was significantly activated in the differentiated podocytes. The phosphorylation status of MLC and ERM, two ROCK downstream substrates, were also enhanced in differentiated cells. Inhibition of ROCK kinase activity diminished both MLC and ERM phosphorylation and disrupted the formation of FA, stress fibers, and cell-cell junctions, indicating the importance of ROCK activity for the maintenance of podocyte structure. ROCK2 gene knockout podocytes were then generated by CRISPR/Cas9 method. Comparing with wild-type cells, ROCK2-/- cells exhibit less central stress fibers, instead of enhanced cortical actin at cell peripheral. The interdigitating cell processes with neighboring cells was also affected. The phosphorylation of MLC, but not ERM, was significantly decreased in ROCK2-/- cells. Taken together, ROCK2 is important for actomyosin contractility that is critical for the maintenance of cell architecture in cultured podocytes.